Microbiome Data Analysis Snakemake Workflows

Integrated Solutions for Better Results

Note: We use the snakemake workflow engine¹ to maintain reproducibility in technical validation, regeneration of results, and improvement of the microbiome bioinformatics analysis.

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Improved Mothur Snakemake Workflows

imap-mothur

• A workflow for microbial profiling using 16S rRNA gene markers.
• We review existing workflows² and create a more reproducible pipeline.
• Each major step form a separate snakemake rule.

Workflow framework

Microbial profiling classification options

1. Operational Taxonomic Units (OTUs)

• OTUs are clusters of similar sequences and are commonly accepted as analytical units in microbial profiling when using 16S rRNA gene markers.

2. Phylotype

• A phylotype in microbiome research is a DNA sequence or group of sequences sharing more than an arbitrarily chosen level of similarity of a 16S rRNA gene marker.

3. Amplicon Sequence Variant (ASV)

• An ASV in microbiome research is any inferred single DNA sequences recovered from a bioinformatics analysis of 16S rRNA marker genes.

4. Microbial Phylogenies

• Microbial phylogenies are from gene sequence homologies. Models of mutation determine the most-likely evolutionary histories.

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Appendix

Reference Databases

1. Mothur-based SILVA reference files³.
2. Mothur-based RDP reference files⁴. Note: The RDP database is to classify 16S rRNA gene sequences to the genus level.
3. ZymoBIOMICS Microbial Community Standard (Cat # D6306)⁵. The ZymoBIOMICS Microbial Community DNA Standard is designed to assess bias, errors and other artifacts after the step of nucleic acid purification.

Sample location (demo)

Troubleshooting (in progress)

1. Mothur dist.seqs taking too long.
• Merged reads are too long, probably over 300pb.
• Reads not overlaping when merging the paired reads.
• Too many uniques representative sequences probably caused by lack of overlapping.
• No enough computer power which suggest a use of HPC or Cluster.

References

1. Köster, J., Mölder, F., Jablonski, K. P., Letcher, B., Hall, M. B., Tomkins-Tinch, C. H., Sochat, V., Forster, J., Lee, S., Twardziok, S. O., Kanitz, A., Wilm, A., Holtgrewe, M., Rahmann, S., & Nahnsen, S. (2021). Sustainable data analysis with snakemake. F1000Research, 10. https://doi.org/10.12688/f1000research.29032.2

2. Close, W. L. (2020). Mothur 16S v4 analysis pipeline. https://github.com/wclose/mothurPipeline

3. Mothur-based silva reference files. https://mothur.org/wiki/silva_reference_files/

4. Mothur-based RDP reference files. https://mothur.org/wiki/rdp_reference_files/

5. ZymoBIOMICS microbial community DNA standard (cat # D6306). https://www.zymoresearch.com/zymobiomics-community-standard

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Last updated on January 25, 2023.

These workflows are continuously reviewed, integrated, tested and compiled by Teresia Mrema-Buza.
Github_Repo: https://github.com/tmbuza/imap-mothur.
Github_Page (this document): https://tmbuza.github.io/imap-mothur/.
Visit the Complex Data Insights (CDI) website for more practical user guides (…in progress).